Outer membrane vesicles from gram negative bacteria and use as a vaccine

ABSTRACT

A composition is prepared from a mixture of different vesicles, such as outer membrane vesicles (OMVs) and vaccines are based thereon. Another composition comprises in a single vesicle a combination of antigens and/or other vesicle components deriving from separate vesicles; again vaccines are prepared therefrom.

The present invention is in the field of compositions comprisingvesicles, such as liposomes and outer membrane vesicles (OMVs)obtainable from Gram negative bacteria, methods of making suchcompositions and vaccines based thereon. In particular, the inventionrelates to vaccines and pharmaceutical compositions comprising OMVsobtained from Neisseria species.

A significant number of human and animal pathogens fall within the Gramnegative classification of bacteria, including members of the genusNeisseia, Moraxella, Kingella, Acinetobacter, Brucella, Bordetella,Haemophilus, Escherichia, Chlamydia, Legionella, Pseudomonas, Proteusand Yersinia. Neisseria meningitidis (the meningococcus) is the organismthat causes meningococcal meningitis and is of particular importance asa worldwide health problem. In many countries the incidence of thisdisease is increasing. N. meningitidis is also responsible formeningococcal septicaemia, which is associated with rapid onset and highmortality, with around 22% of cases proving fatal. Other Gram negativebacteria are responsible for a range of human infections includingmeningitis (H. influenzae), plague (Y. pestis), gastroenteritis (E.coli), venereal disease (N. gonorrhoeae) and nosocomial infection (P.aeruginosa).

It would be desirable to provide alternative broad spectrum vaccinesthat provide protective immunity in animals, particularly humans,against Gram negative bacterial infection, and especially infection byGram negative pathogens.

Many known vaccines are based upon preparations of capsularpolysaccharide, however, these vaccines are often limited in theirprotective value. For example, vaccines directed at providing protectiveimmunity against meningococcal disease provide only limited protectionbecause the protection tends to be strain specific whereas there aremany different strains of N. meningitidis. Vaccines based upon theserogroup antigens, the capsular polysaccharides, offer only short livedprotection against infection and do not protect against many strainscommonly found in North America and Europe. In fact, certain capsularpolysaccharides, such as that from the group B meningococcal capsule,are essentially non-immunogenic in humans.

The outer membrane of many Gram negative bacteria is highly dynamic andcan produce vesicles that bud off and are released into the surroundingenvironment. These outer membrane vesicles (OMVs), also referred to asblebs, comprise many of the outer membrane proteins (OMPs) andlipopolysaccharide (LPS) that contribute to the antigenic profile of theorganism.

There have been a number of attempts to generate an OMV based vaccine inthe hope that it could overcome the disadvantages seen in previouscapsular polysaccharide based vaccines. In Bjune et al. (Lancet (1991)338: pp1093-1096) a vaccine consisting of OMVs from group B N.meningitidis is described (Norwegian vaccine). Bjune et al. show thatthe vaccine was able to induce a protective efficacy againstmeningococcal disease of 57.2% in a clinical trial in Norway. A similarvaccine has been produced in Cuba (Sierra et al., NIPH Ann (1991)December;14(2): pp195-207) and high levels of efficacy were observed inthat country. However, a large study in Brazil showed poor efficacy ofthe Cuban vaccine, especially in young children (de Moraes et al.,Lancet (1992) October 31;340(8827): pp1074-1078).

To address the difficulties associated with achieving broad spectrumprotection researchers have attempted to “enrich” OMVs with particularantigens that might enhance the immunogenic potential of the OMV. InWO-A-00/25811 OMVs isolated from N. meningitidis are combined withheterologous antigens, e.g. Tbp, or a genetically modified N.meningitidis expresses such antigens recombinantly and antigen enrichedOMVs are derived therefrom. A similar approach was adopted byresearchers in WO-A-01/09350 which describes vaccine compositionscomprising OMVs from N. meningitidis, M. catarrhalis and H. influenzae,where in certain embodiments these organisms have been geneticallymodified to overexpress particular immunogenic moieties.

A further OMV based vaccine composition is known as the Hexamen™ orDutch vaccine (Cartwright et al., Vaccine 17 (1999), pp2612-2619). TheHexamen™ vaccine composition comprises N. meningitidis OMVs that includesix different PorA proteins that are recombinantly produced using twovaccine strains of N. meningitidis, PL16215 and PL10124. Each strain iscapsule negative and produces three different PorA proteins,CPS⁻P1.7,16;P1.5,2; P1.19,15 and CPS⁻P1.5^(c),10; P1.12,13; P1.7^(h),4respectively.

Ruppe Van der Voort et al (Vaccine (2000) 18(14); pp 1334-1343) showthat the hexamen vaccine induces specific serum bactericidal antibodiesagainst all six PorA sero-subtypes included in the vaccine. However, theHexamen™ vaccine suffers from certain drawbacks. It is not currentlypossible to express all six PorA proteins in a single N. meningitidiscell due to host toxicity problems. Hence, three PorAs are expressed inone cell and three in another. OMVs from cell cultures each expressingthree PorAs are obtained and then admixed to give the hexavalentvaccine.

PorA is an immunodominant antigen, meaning that it masks most otherantigens presented on the OMV surface—i.e. Hexamen™ vaccine is heavilybiased towards PorA as the protective antigen. This, in turn, leads toselection pressure in the population towards strains of N. meningitidisthat express PorAs that are antigenically different to those in theHexamen™ vaccine. There is, therefore, a risk that strains of N.meningitidis that are not protected against by the Hexamen™ vaccine willpredominate in time, resulting in ongoing efforts to continually modifythe vaccine to protect against strains currently infecting and causingdisease in the human population.

It would be desirable to provide an OMV vaccine composition thatprovides broad spectrum protection to infection from a number ofbacterial species and at least a wide range of strains within a singlebacterial genus. It would be particularly desirable to produce anOMV-based vaccine that provides broad spectrum, long term protectionagainst disease caused by a range of strains of Gram negative bacteria,and especially N. meningitidis.

It would further be desirable to provide further and/or improved methodsof preparing vesicle-containing compositions.

Accordingly, the present invention provides methods and compositionscomprising vesicles, especially OMVs, and vaccine compositions basedthereon which can provide a broad scope of protection to infection fromGram negative bacterial pathogens, such as N. meningitidis.

An advantage of compositions and vaccines of the present invention isthat the OMVs are derived from a diversity of Gram negative bacterialsources, thus allowing the composition to present a broad spectrum ofantigens to the host immune system, and thereby generating broadspectrum protective immunity.

The present invention also provides methods of combining vesicles,especially OMVs, vesicles obtained thereby and vaccine compositionsbased thereon. Advantages of the combining include control of vesiclecontent and/or facilitation of antigen combinations in the resultantvesicles.

In order to facilitate understanding of the present invention a numberof terms used herein are defined in more detail below.

Gram negative bacteria are those bacteria that fail to resistdecolourisation in the commonly known Gram staining method. Gramnegative bacteria are characterised by a complex multilayer cell walland often possess an outer layer polysaccharide capsule—e.g. N.meningitidis, although in some species this capsule is absent—e.g. N.lactamica.

The term pathogenic as used herein refers to an organism that is capableof causing disease, particularly in animals and especially in humans.

The term “non-pathogenic” refers to organisms that do not cause diseasein animals, in particular in humans. The term includes commensalorganisms. Commensal organisms are those that can colonize a hostorganism without signs of disease. Examples of commensal organismsinclude the commensal Neisseria species, such as N. lactamica, N. sicca,N. cinerea, N. perflava, N. subflava, N. elongata, N. flavescens, and N.polysaccharea.

Outer membrane vesicles (OMVs), also referred to as blebs, are vesiclesformed or derived from fragments of the outer membrane of a Gramnegative bacterium. OMVs typically comprise outer membrane proteins(OMPs), lipids, phospholipids, periplasmic material andlipopolysaccharide (LPS). Gram negative bacteria, especially pathogenslike N. meningitidis, often shed OMVs during virulent infections in aprocess known as blebbing. OMVs can also be obtained from Gram negativebacteria via a number of chemical denaturation processes described inmore detail in the Examples below. Liposomes, comprising a lipid bilayerand typically enclosing an aqueous core, can be regarded for thepurposes of the present invention as constituting a synthetic equivalentto OMVs, and embodiments of the invention described with reference toOMVs apply mutatis mutandis to embodiments carried out with and relatingto liposomes. A distinction between liposomes and OMVs may be made forexample in embodiments in which control of content of a liposome ispossible whereas OMV content is not so readily controllable.

A “vaccine” as referred to herein is defined as a pharmaceutical ortherapeutic composition used to inoculate an animal in order to immunizethe animal against infection by an organism, typically a pathogenicorganism. A vaccine will typically comprise one or more antigens derivedfrom one or more organisms which on administration to an animal willstimulate active immunity and protect that animal against infection withthese or related pathogenic organisms.

Vaccine compositions that are formulated as pharmaceuticals willtypically comprise a carrier. If in solution or in liquid aerosolsuspension, suitable carriers can include saline solution, sucrosesolution, or other pharmaceutically acceptable buffer solutions. Anaerosol formulation will typically additionally comprise a surfactant.Alternative vaccine compositions include microencapsulated OMVcompositions. Such microcapsules with generally comprise a biocompatiblepolymer shell or core, such as made from polylactide-co-glycolide (PLG).Vaccine compositions can additionally comprise an adjuvant, for examplewhere administration is via the parenteral route. Suitable adjuvantsinclude aluminium hydroxide.

Vaccines are suitably administered to an animal via a number routes. Forexample, parenterally—e.g intramuscularly, trans-dermally—or via otherroutes—e.g. intra-nasally, orally, topically—or via any other commonlyknown administrative route.

Certain proteins can be recombinantly expressed in Gram negativebacteria and thereby enable enrichment or alteration of the antigenicprofile of the bacterial outer membrane. Genetic modification of abacterial source organism thereby allows for manipulation of theantigenic profile of OMVs that are obtained from these organisms. Whenproteins that are not normally present in the bacterial outer membrane,and thus in an OMV derived therefrom, are introduced via recombinantexpression techniques, these “non-native” proteins and polypeptides aredescribed as heterologous antigens. The contents of WO-A-00/25811 andWO-A-01/09350 are incorporated herein. Thus it is an advantage of theinvention that the vaccine comprises OMVs rather than live attenuated ordead pathogenic organisms which can pose a greater risk of infection ortoxicity.

A first aspect of the invention mixes different vesicle preparations.This may be done to alter the immunogenicity of a first OMV preparationor to mix useful properties in respective preparations.

The invention thereby provides compositions comprising vesicles derivedfrom two or more sources. The vesicles are preferably lipid vesiclescomprising a lipid bilayer surrounding an aqueous core. Typically thelipid vesicles are of unilamellar structure (i.e. a single lipid bilayersurrounds the aqueous core), although multilammellar lipid vesicles arealso suitably used in the compositions of the invention.

These lipid vesicles are preferably synthetic vesicles such as liposomesor obtained from bacteria such as outer membrane vesicles (OMVs) byextraction of naturally occurring OMVs (N-OMVs) or using a detergentextraction (D-OMVs). They typically have sizes in the nanomolar tomicromolar range, e.g. from 1 nm to 100 μM, more typically from 10 nm to10 μm and preferably from 30 nm to 1 μm. Antigenic components can belocated in any or all of the three main compartments of the lipidvesicle, namely:

-   -   1. attached to the either the interior or exterior surface of        the lipid vesicle, for example via a membrane anchor domain, or        attachment to a lipid moiety;    -   2. inserted into the lipid bilayer, for example where the        antigenic component is itself a hydrophobic or lipid based        entity; or    -   3. located within the aqueous centre/core of the lipid vesicle.

Where liposomes are utilised in the compositions and methods of theinvention these can typically contain a number of different lipids andfatty acids. Suitable lipids for inclusion in liposomes of the inventioninclude but are not limited to phosphatidylinositol-(4,5)-diphosphate,phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine,phosphatidylglycerol, cholesterol, beta-oleolyl-gamma-palmitoyl, certainlipopolysaccharides and galactocebrosides. Liposomes can be obtainedcommercially from a number of sources and methods for preparingliposomes suitable for use in the invention are known in the art.

The lipid vesicles for use in the invention can be enriched and/orsupplemented with preferred antigenic components. Regimes forsupplementing the lipid vesicles, whether they be liposomes or OMVs,include via direct combination in vitro where an energetic combinationstep can optionally be applied to facilitate integration of theantigenic component into one or more of the three aforementioned lipidvesicle compartments. Preferred, energetic combination methods includehomogenisation, ultrasonication, extrusion and combinations thereof.

One advantage associated with liposomes is that the exact antigeniccomposition of the lipid vesicle can be controlled and batch to batchquality control maintained more easily than with lipid vesicles derivedfrom biological sources. In addition, the physical properties of theliposome, such as size, are more easily controlled by modifying thelipid composition of the bilayer.

However, certain antigenic components are difficult to synthesise invitro, or to isolate in pure form from biological sources. Further,certain antigenic components are difficult to integrate in antigenicallyactive form into synthetic liposomes. Hence, in such cases OMVs are thepreferred lipid vesicle. Further advantages of using OMVs are thatantigenic components can be synthesised in the host organism from whichthe OMV is obtained thereby providing a convenient method forintegrating a preferred antigenic component into a lipid vesicle.

In situations where a particular component is not desired in a lipidvesicle of the invention, for example endotoxin, different options applywhen using liposomes or OMVs. It is apparent that as the composition ofthe liposome is synthetically controlled the non-desired component issimply not added to the liposome. This represents a further advantageassociated with the use of liposomes. Where OMVs are preferred as thelipid vesicle, the OMVs can be obtained from organisms that arenaturally free or low in the non-desired component, for examplecommensal organisms. Alternatively, knock out organisms can be generatedwhere genes coding for or regulating the production of the non-desiredcomponent are deleted or silenced.

The lipid vesicles for use in the invention can also comprise abiologically active component, other than an antigen, within the aqueouscentre/core. This component is suitably selected from soluble adjuvants,cytokines, immunomodulatory agents, pharmaceuticals, excipients,proteins, polypeptides and pharmacologically or pharmaceutically activeagents.

The invention also provides a composition comprising OMVs obtained fromat least two different species of Gram negative bacteria, wherein atleast one of said species is a non-pathogenic species. In general, allsuch bacteria are believed suitable, though Gram negative speciesespecially suitable for use in the invention include those selected fromNeisseria, Moraxella, Kingella, Acinetobacter, Brucella, Bordetella,Porphyromonas, Actinobacillus, Borelia, Seffatia, Campylobacter,Helicobacter, Haemophilus, Escherichia, Legionella, Salmonella,Pseudomonas and Yersinia. In a particular embodiment of the inventionthe composition comprises a pathogenic species is selected from strainsof N. meningitidis.

The non-pathogenic species can suitably be any non-pathogenic Gramnegative species. In a specific embodiment of the invention described inmore detail below, the composition comprises OMVs obtained from acommensal Neisseria.

Compositions of the invention need not be limited to comprising OMVsfrom only two sources. The invention also provides compositionscomprising OMVs from a plurality of Gram negative bacterial sources,from as few as three sources up to many tens or more. It is feasible fora library of a multiplicity of OMVs to be constructed and hence certaincombinations of OMVs can be formulated into compositions, according toprecise requirements, so as to provide a particular predeterminedspectrum of vaccination coverage. For example, where a certain strain ofpathogen is prevalent in a particular geographical area, OMVs specificto this pathogen are incorporated into a broad spectrum vaccinecomposition in order to provide improved local efficacy.

Combining a plurality of OMVs in a single vaccine composition provides acomposition capable of conferring broad spectrum immunity and alsoenables the “dilution” of immunodominant antigens, such as PorA, whichwould otherwise mask the other antigens present in the composition.

Another composition of the invention comprises OMVs, which OMVs areobtained from a first and at least a second different species ofNeisseria, wherein:

-   -   1. the first species is a pathogenic species of Neisseria; and    -   2. the at least a second species is a commensal species of        Neisseria.

In a specific embodiment of the invention, the composition comprisesOMVs obtained from a first and at least a second different species ofNeisseria, wherein:

-   -   1. the first species is selected from the group comprising N.        meningitidis and N. gonorrhoeae; and    -   2. the at least a second species is selected from the group        comprising N. lactamica, N. sicca, N. cinerea, N. perflava, N.        subflava, N. elongata, N. flavescens, and N. polysaccharea.

In a further specific embodiment of the invention, some or all of theOMVs in the composition also comprise a heterologous antigen. Suitableheterologous antigens include cell membrane associated proteins, orperiplasmic proteins, such as PorA; Cu—Zn-SOD; TspA, LbpA, LbpB, pilQ,TbpA; TbpB and/or NspA. These can also be non-neisserial, such as B.pertussis toxin, diphtheria toxin, tetanus toxin, measles antigen, HIVantigens, smallpox antigen, and/or anthrax antigen. It should be notedthat the OMVs of the invention provide advantageous presentation ofantigens to the host immune system, thereby enabling broad spectrum,long term protective immunity.

A third composition of the invention is one comprising OMVs, which OMVsare obtained from a first neisserial source and at least a secondneisserial source different from the first. The term “source” is used torefer to the genus and/or strain of Neisseria from which the OMVs areobtained or isolated. Hence, if OMVs are extracted from N. meningitidisstrain K454, this is the neisserial source of the OMVs.

In one embodiment of the invention the first neisserial source is acommensal Neisseria and said at least second neisserial source is apathogenic Neisseria. Alternatively, the first neisserial source can bea commensal Neisseria and said at least second neisserial source is alsoa commensal Neisseria but of a different species or strain to the first.For example, the first source is suitably N. lactamica and the second N.cinerea. In a second example, the first source is N. lactamica strainY921009 and the second source is N. lactamica strain 2086. A furtheroption is to utilise different genetic mutants of the same strain.

As mentioned previously, the invention also utilizes OMV compositionsderived from neisserial sources which are genetically modified so as torecombinantly express one or more heterologous antigens. This can beachieved by transforming the cells with an expression vector thatcomprises DNA encoding the desired antigenic polypeptide. Alternatively,an endogenous polypeptide can be upregulated or modified in some way asto be expressed either on the surface or within the periplasm of thecell, and thereby incorporated into OMVs extracted from that cell.Although such proteins are endogenous to the cell, their presence atabnormal levels in OMVs, can still be considered to fall within thescope of the term “heterologous antigen”.

In a particular embodiment of the invention a first neisserial source isa commensal Neisseria that expresses a first heterologous antigen andthe second neisserial source is a Neisseria that expresses a secondheterologous antigen different from the first antigen. In a furtherembodiment of the invention a first neisserial source is a commensalNeisseria that expresses a first heterologous antigen and the least asecond neisserial source is a commensal Neisseria of the same or asimilar strain that expresses a second heterologous antigen differentfrom the first antigen. Each source can further optionally expressmultiple heterologous antigens.

A fourth composition of the invention comprises an OMV containing outermembrane protein (OMP) and/or lipopolysaccharide (LPS) derived from atleast two different species of Gram negative bacteria, such asNeisseria. In specific embodiments of the invention, the OMV is suitablyderived from two species of Neisseria which include a commensalNeisseria and a pathogenic Neisseria, from at least two species ofcommensal Neisseria, or even from two different strains of the samecommensal species. The OMV can optionally further comprise one or morerecombinantly expressed polypeptides.

It is an advantage of the present invention that the OMVs derived fromcommensals typically have LPS of lower toxicity than the LPS found inthe outer membrane of pathogenic species. Hence, vaccine compositionscomprising commensal OMVs typically elicit lesser adverse reactions thancompositions comprising pathogenic OMVs.

The LPS content of the OMV, however, also provides an adjuvant effectwhich itself enhances an immune response especially in compositions thatare administered intra-nasally. It is a further option to derive OMVsfrom LPS null mutant or LPS modified species of Gram negative bacteria,suitably in cases where potential LPS toxicity is likely to causeextreme allergic reactions.

OMVs of the invention can be suitably mixed from sources such as Gramnegative bacteria of different species or strains. Alternatively, OMVscan optionally be obtained from sources that are from the same commensalstrain but where each source expresses different heterologous antigens.A further example of different OMVs from same strain sources is whereOMVs are obtained from a Gram negative bacterial source at differentphases in the organism's growth cycle. The OMVs are then combined into asingle composition that represents the surface antigenicity profile ofthe organism throughout its growth cycle.

A second aspect of the invention combines different vesicle preparationsso as to transfer a component of one vesicle, or one type of vesicle, toanother. By way of example, liposomes are combined with liposomes,liposomes with OMVs and OMVs with OMVs.

The invention thus provides a method of preparing a composition,comprising:—

-   -   1. obtaining a first composition which contains first vesicles,        said first vesicles having a first antigenic component;    -   2. obtaining a second composition which contains second        vesicles, said second vesicles having a second antigenic        component different from the first antigenic component; and    -   3. combining the first and second compositions so as to obtain a        third vesicles-containing composition, wherein the third        vesicles-containing composition comprises third vesicles having        both the first antigenic component and the second antigenic        component.

A further method of the second aspect, for preparing lipid vesicles,comprises:—

-   -   a. synthesising liposomes comprising a first antigenic        component;    -   b. obtaining OMVs from an organism, said OMVs comprising a        second antigenic component; and    -   c. mixing the liposomes of (a) with the OMVs of (b) so as to        form a vesicle comprising both the first and the second        antigenic component.

A still further method of the second aspect, for preparing an OMVcontaining composition, comprises:—

-   -   1. isolating OMVs from a first species of Gram negative        bacteria, wherein said first species is either pathogenic or        non-pathogenic to humans;    -   2. isolating OMVs from at least a second species of Gram        negative bacteria different from the first, wherein said second        species is non-pathogenic to humans;    -   3. combining the OMVs from (1) and (2) to form a hybrid OMV        containing at least a portion of an OMV from step 1 and at least        a portion of an OMV from step 2.

The second aspect of the invention extends also to compositionsobtainable and obtained using the above methods, to pharmaceuticalcompositions, to methods of medical treatment as herein described, touses as herein described, all based thereon.

In use of methods of this aspect of the invention, the combining of thetwo separate compositions, containing distinct vesicles, is carried outso as to effect a transfer of an antigen between the respectivecompositions, resulting in production of a third type of vesicle whichcontains antigenic components derived from both the first and the secondoriginal vesicles. An advantage of this combination is that there isprovided as a result a vesicle containing both first and secondantigenic components, and this can be purified so as to provide ahomogenous preparation of vesicles containing both such antigeniccomponents.

This means of providing a vesicle with both antigenic components can beeasier than, for example, carrying out a transfection of a bacteria sothat it expresses both antigenic components. In addition, as can beappreciated, homogenous preparation of many different combinations ofantigens can be prepared from starting materials comprising individualvesicles containing individual antigenic components of interest.Different antigens may be expressed at different levels in the samebacterial host. Using the above methods of combination of vesicles, bycontrol of the starting amount/concentration of each respective vesicle,with its respective antigen, the relative amounts/concentrations ofantigens in the final resultant combined vesicle can be controlled.

It is preferred to separate the third vesicles from thethird-vesicles-containing composition, to obtain a purified preparationof the third vesicles. This can be carried out using a double-immunemethod. Thus, a first purification is carried out with an antibody (e.g.immobilized), specific for the first antigen; a second purification isthen carried out using an antibody specific for the second antigen—thispurifies the composition in respect of vesicles containing bothantigens.

Both OMVs and liposomes, and combinations thereof are suitable for themethods of the second aspect, with OMVs preferably being derived fromGram negative bacteria as previously described—though especially fromNeisseria.

In a development of the second aspect of the invention, a liposomecontaining a component other than (or in addition to) an antigen can becombined with an antigen-containing vesicle.

Thus, one such method of preparing a composition, comprises:—

-   -   1. obtaining a first composition which contains first vesicles,        said vesicles having an antigenic component;    -   2. obtaining a second composition which contains second        vesicles, said second vesicles comprising a soluble,        biologically active component within aqueous cores of the        vesicles; and    -   3. combining the first and second compositions so as to obtain a        third vesicles-containing composition, wherein the third        vesicles-containing composition comprises third vesicles which        both comprise the antigenic component and also contain, within        an aqueous core, the soluble, biologically active component.

These resultant vesicles confer the advantage of combining, at thecontrol of the user, the antigen from one source and the liposomecontents of another, resulting in provision of further methods forpreparation of vesicles for vaccination and other uses.

In a preferred composition of the invention liposomes comprising one ormore antigenic components are combined with OMVs in a singlecomposition. Following a fusion event in which a modified energeticcombination step is employed, resulting hybrid or chimaeric lipidvesicles are formed.

In a further preferred composition of the invention liposomes comprisingone or more antigenic components are combined with OMVs in a singlecomposition so that antigens from OMVs are exchanged or transferred toliposomes and vice versa. After the antigen exchange step, the liposomesand OMVs can be separated (for example, by centrifugation) and formdistinct compositions in their own right for application as vaccinecompositions as described further herein.

A further aspect of the invention provides an OMV composition,characterised in that each OMV in said composition comprises OMP and LPSfrom at least two different species of Neisseria. Optionally, at leastone of the species of Neisseria is a commensal Neisseria. It is furtheroptional for one of the species to be a pathogenic species of Neisseria,for example N. meningitidis or N. gonorrhoeae.

In further examples of the invention, there are provided methods ofpreparing an OMV composition comprising the steps of:

-   -   1. obtaining OMVs from a first species of Gram negative        bacteria, wherein said first species is either pathogenic or        non-pathogenic to humans;    -   2. obtaining OMVs from at least a second species of Gram        negative bacteria different from the first, wherein said second        species is non-pathogenic to humans; and    -   3. combining the OMVs from (1) and (2).

Also provided is a method for preparing a vaccine composition, whichmethod is substantially identical to the above-mentioned method, butwhich instead comprises the step of:

-   -   3. combining the OMVs from (1) and (2) together with a        pharmaceutically acceptable carrier.

Suitable methods for extracting OMVs from bacterial sources includedeoxycholate extraction, Tris/HCl/EDTA extraction, and lithium acetateextraction. Protocols for performing such extractions are described inmore detail in the Examples below. However, it will be appreciated bythe skilled person that virtually any chemical and/or physical techniquethat enables disruption of the bacterial cell outer membrane in order torelease sufficient OMVs for purification and isolation, would besuitable for preparation of the compositions of the invention

In a specific embodiment of the invention, combined OMVs are homogenisedin a low power homogeniser (e.g. Waring blender or Silverson homogeniseror by ultrasonication). This additional step has the effect ofdisrupting the OMVs in the mixture such that they fuse to form hybridOMVs. In this way a single OMV can comprise OMPs, LPS and heterologousantigens from a plurality of bacterial sources. The fused chimaeric OMVsadvantageously enable the presentation of multiple antigens to a hostimmune system in a uniquely immuno-available form.

Further aspects of the invention provide methods of vaccinating animals,especially humans, against Gram negative bacterial infection utilisingthe compositions of the invention. In particular, the invention providesmethods for vaccinating animals against meningococcal infection. Alsoprovided are uses of the compositions of the invention in thevaccination of animals, including humans, against Gram negativebacterial infection. Further provided are uses of the compositions ofthe invention in the manufacture of vaccines for inoculating animals inorder to stimulate protective immunity to Gram negative bacterialinfection. OMVs are of use in mucosally administered compositions, asLPS toxicity is less and LPS can function as an adjuvant.

The invention is now described in specific examples with reference tothe accompanying drawings in which:—

FIG. 1 shows an electron micrograph of a preparation of N. meningitidisOMVs;

FIG. 2 shows an electron micrograph of a preparation of N. lactamicaOMVs; and

FIG. 3 shows an electron micrograph of a blend of N. meningitidis and N.lactamica OMVs according to the invention.

EXAMPLES Example 1

Deoxycholate Extraction to Produce OMVs

This method is based on that used to produce the Norwegian Institute ofPublic Health OMV vaccine for parenteral delivery (Fredriksen, J H etal. (1991) Production and characterisation of menB-vaccine “Folkehelsa”:an outer membrane vesicle vaccine against group B meningococcal disease.NIPH Annals 14 (2): 67B 80).

Reagents:

-   Frantz medium-   Buffer 1: 0.1M Tris-HCl pH8.6 containing 10 mM EDTA, 0.5% (w/v)    deoxycholate (DOC), 0.01% (w/v) thiomersal.-   Buffer 2: 50 mM Tris-HCl pH8.6 containing 2 mM EDTA, 1.2% (w/v) DOC,    20% (w/v) sucrose, 0.01% (w/v) thiomersal-   Buffer 3: 50 mM Tris-HCl pH8.6 containing 3% (w/v) sucrose, 0.01%    (w/v) thiomersal    Method-   1. Appropriate N. meningitidis or commensal Neisseria strains were    grown in Frantz medium at 37° C. with shaking until cultures had    reached early stationary phase.-   2. Once cultures reached early stationary phase the culture was    stored overnight at 4-8° C.-   3. The culture was harvested by centrifugation, 5000×g for 15 min at    4° C.-   4. To the pellet, buffer 1 was added using a ratio of buffer to    biomass of 5: 1 (v/w).-   5. The suspension was centrifuged at 20,000×g for 30 min at 4° C.    and the supernatant retained.-   6. The extraction was repeated with 0.1 M Tris buffer with the    volume reduced to one third of that used in step 4. Again, the    supernatant was retained.-   7. The supernatant from steps 5 and 6 was pooled and    ultracentrifuged at 100,000×g for 2h at 4° C.-   8. The resultant OMV pellet was resuspended in buffer 2.-   9. Ultracentrifugation was repeated as in step 7.-   10. OMVs were then homogenised in buffer 3.-   11. OMV preparation was stored at 4-8° C.

Example 2

Tris-HCl/EDTA Extraction to Produce NOMVs (Native OMVs)

This method is based on that used by N. B. Saunders et al.(Immunogenicity of intranasally Administered Meningococcal Native OuterMembrane Vesicles in Mice. Infection and Immunity (1999) 67 (1):p.113-119). OMVs prepared in this way have been used as an intra-nasalvaccine in human volunteers.

Reagent:

-   Stock NOMV buffer: 0.15M NaCl, 0.05M Tris-HCl, 0.01 M EDTA pH 7.5.    Method:-   1. The culture (Fe limited) was prepared in 500 ml Frantz medium per    strain.-   2. Cells from the 500 ml culture in step 1 were centrifuged at 3500    rpm for 15 minutes.-   3. Cells were resuspended in 25 ml NOMV buffer.-   4. The suspension was warmed at 56° C. for 30 minutes.-   5. The suspension was sheared in Waring blender for 3 minutes (low    speed).-   6. The suspension was centrifuged at 23,500 g for 20 minutes.-   7. The supernatant was retained.-   8. The pellet was resuspended in 12 ml distilled water and    centrifuged at 23,500 g for 20 minutes.-   9. The supernatant was retained and combined with supernatant from    step 7.-   10. The supernatants were centrifuged at 23,500 g for 20 minutes.-   11. The supernatant was retained and centrifuged at 100,000 g for 2    hours-   12. The pellet was washed by repelleting from distilled water.-   13. Resulting NOMVs were stored in PBS at 4° C.

Example 3

Lithium Acetate Extraction to Produce OMVs

This is a further alternative method of producing OMVs (Hamel, J. et al.1987. J. Med. Microbiol. 23,163-170).

Reagent:

-   Lithium acetate buffer:−200 mM Lithium acetate+5 mM EDTA pH 6.0    Method:-   1. The broth culture (Fe limited) was prepared in 100 ml MHB per    strain.-   2. Cells from 100 ml culture were centrifuged at 3500 rpm for 15    minutes.-   3. Cells were resuspended in 20 ml Lithium acetate buffer (LiAc)(for    750 ml cultures, resuspend cell pellet in 30 ml LiAc)-   4. The suspension was incubated for 3h at 37° C. with shaking (180    rpm).-   5. The cell suspension was then passed through a 21 gauge needle 7    times, alternatively a bead beater can be used.-   6. The suspension was centrifuged at 16000 rpm for 20 minutes.-   7. The supernatant was carefully recovered.-   8. The supernatant was centrifuged at 35,000 rpm for 2h and the    pellet collected.-   9. The pellet was resuspended in PBS and stored at 4° C.

Example 4

Alternative Deoxycholate Extraction Method for Production of an OMVBased Vaccine Suitable for Parenteral Administration

-   1. Strains were cultured in Frantz medium (135 I) to early    stationary phase; cells were harvested by continuous flow    centrifugation and resuspended in NaCl buffer.-   2. The cell suspension was homogenised for 30 min and the total wet    weight of the suspension determined.-   3. The cell suspension was centrifuged for 60 min at 2900×g and the    pellet resuspended in 0.1 M Tris-10 mM EDTA buffer at a ratio of    7.5:1 wet weight-   4. Extraction of the vesicles was performed by the addition of    {fraction (1/20)}^(th) volume of 0.1 M Tris, 10 mM EDTA, 10%    deoxycholate (DOC).-   5. Vesicles were separated from cell debris at 20000×g at 4° C. for    1 h-   6. The supernatant containing the vesicles was concentrated by    ultracentrifugation at 125000×g at 4° C. for 2h.-   7. The OMV pellet was resuspended in 0.1 M Tris, 10 Mm EDTA, 0.5%    DOC buffer and the suspension centrifuged again at 125000×g at 4° C.    for 2h.-   8. The concentrated OMVs were resuspended in 3% sucrose solution-   9. To prepare the adjuvanted vaccine, OMV extracts were mixed in    equimolar amounts with AlPO₄ as adjuvant.

Example 5

Blended OMV Vaccine Composition

Separate OMV extracts prepared according to any of the methods describedin Examples 14 are obtained from N. meningitidis and N. lactamica. TheOMV extracts are combined as per step 9 in Example 4.

Example 6

Blended OMV Vaccine Composition

N. meningitidis and N. lactamica cultures are mixed together prior toperforming the OMV extraction methods described in Examples 1-4.Concentrated OMVs are resuspended in 3% sucrose solution.

Example 7

Blended OMV Composition

OMV extracts are obtained from N. meningitidis and N. lactamicaaccording to any of the methods given in Examples 1-4. Extracts of eachare combined into a single composition and then homogenized.Concentrated OMVs are resuspended in 3% sucrose solution.

Example 8

Blended OMVs Composition

Reagents

-   N. lactamica OMVs from strain Y92 1009-   N. meningitidis OMVs from strain MC 58 (B:15:P1.7,16)-   Anti-meningococcal serosubtype (PorA) P1.7 monoclonal antibody    (95/706 from NIBSC, UK)-   AffiniPure anti-mouse IgG (Jackson Immuno Research Laboratories) 6    nm colloidal gold conjugate-   Blocking buffer (2% bovine serum albumin in PBS)-   Conjugate buffer (0.02% Tween 20, 0.1% BSA, 5% newborn calf serum in    PBS)-   1% potassium phosphotungstate (PTA) stain    Method

N. lactamica (NL) and N. meningitidis (NM) OMVs were removed fromstorage at −20° C. and allowed to thaw at room temperature. A mixture ofOMVs was prepared separately by adding equal volumes of NL and NM OMVsto a glass container and agitating gently using a pipette; all OMVs werethen stored at 4° C. until required.

NM, NL and the mixture of OMVs were placed on 3 separate carbon coatedcopper grids and allowed to dry. The grids were then placed in 100 μl ofa 1:500 dilution of meningococcal PorA monoclonal antibody and incubatedat room temperature for 2h. The grids were washed twice in blockingbuffer (PBS containing 2% BSA) and added to a 1:20 dilution of 6 nmcolloidal gold particles conjugated to AffiniPure anti-mouse IgG(Jackson Immuno Research Laboratories) in 0.02% Tween 20, 0.1% BSA, 5%newborn calf serum in PBS.

Following 1 h incubation at room temperature the grids were washed twicein blocking buffer, once in distilled water and stained with 1%potassium phosphotungstate (PTA) for 5-10 sec. Grids were then examinedby electron microscopy and are shown in FIG. 1-3.

Black dots indicate labeling with the 6 nm gold particles in FIGS. 1, 2and 3. In FIG. 1, NM OMVs are evenly labeled with the 6 nm goldparticles, showing that PorA is present on all OMVs. In FIG. 2, there isno labeling, consistent with the fact that NL OMVs do not contain PorA(a pathogen—specific antigen not seen in commensal Neisseria). The FIG.3 result, showing the combination of NM and NL OMVs, shows OMVs againevenly labeled with the gold particles. The labeling is even, and isover all OMVs, not restricted to just a fraction of the OMVs. Thus,while half the OMVs in the mixture originally contained no PorA, afterblending all OMVs stain positively for PorA, indicating all OMVs nowcontain PorA.

Thus, the invention provides vesicle-containing, especially OMV-based,preparations and methods for their production.

1-58. (canceled)
 59. A composition comprising outer membrane vesicles(OMVs) obtained from a first and a second species of Neisseria, whereinthe first species is Neisseria meningitidis and the second species is acommensal Neisseria.
 60. The composition of claim 59, further comprisinga pharmaceutically acceptable carrier.
 61. The composition of claim 59,further comprising OMVs obtained from a third species of Neisseria. 62.The composition of claim 59, wherein either or both of said first andsaid second species also comprises a heterologous antigen.
 63. An OMVcomprising outer membrane protein (OMP) and lipopolysaccharide (LPS)from at least two species of commensal Neisseria.
 64. An OMV comprisingOMP and LPS from at least two different strains of commensal Neisseria.65. A method of vaccinating an animal against Gram negative bacterialinfection comprising administering an effective dose of a compositionaccording to claim
 59. 66. A method of treating Gram negative bacterialinfection in an animal comprising administering an effective dose of acomposition according to claim
 59. 67. The method of claim 65, whereinsaid Gram negative bacterial infection is meningococcal meningitis orsepticaemia.
 68. The method of claim 66, wherein said Gram negativebacterial infection is meningococcal meningitis or septicaemia.
 69. Themethod of claim 65, wherein the animal is a human.
 70. The method ofclaim 66, wherein the animal is a human.
 71. The method of claim 65,wherein the effective dose is administered parenterally.
 72. The methodof claim 66, wherein the effective dose is administered parenterally.73. The method of claim 65, wherein the effective dose is administeredintranasally.
 74. The method of claim 66, wherein the effective dose isadministered intranasally.
 75. A method of preparing an OMV compositioncomprising the steps of: 1) obtaining OMVs from a first species of Gramnegative bacteria, wherein said first species is either pathogenic ornon-pathogenic to humans; 2) obtaining OMVs from at least a secondspecies of Gram negative bacteria different from the first, wherein saidsecond species is non-pathogenic to humans; and 3) combining the OMVsfrom (1) and (2).
 76. A method of preparing a vaccine comprising thesteps of: 1) obtaining OMVs from a first species of Gram negativebacteria, wherein said first species is either pathogenic ornon-pathogenic to humans; 2) obtaining OMVs from at least a secondspecies of Gram negative bacteria different from the first, wherein saidsecond species is non-pathogenic to humans; and 3) combining the OMVsfrom (1) and (2) together with a pharmaceutically acceptable carrier.77. The method of claim 75, wherein said first species of Gram negativebacteria is N. meningitidis.
 78. The method of claim 76, wherein saidfirst species of Gram negative bacteria is N. meningitidis.
 79. The Themethod of claim 75, wherein said first and/or at least second species ofGram negative bacteria is a commensal Neisseria.
 80. The method of claim76, wherein said first and/or at least second species of Gram negativebacteria is a commensal Neisseria.
 81. The method of claim 75, whereineither or both of the first and at least second species of Gram negativebacteria comprises a heterologous polypeptide.
 82. The method of claim76, wherein either or both of the first and at least second species ofGram negative bacteria comprises a heterologous polypeptide.
 83. Acomposition, comprising: 1) liposomes obtained from a first source,comprising a first antigenic component; and 2) OMVs obtained from Gramnegative bacteria, comprising a second antigenic component.
 84. Acomposition comprising lipid vesicles, obtainable by: 1) synthesizingliposomes comprising a first antigenic component; 2) obtaining OMVs fromGram negative bacteria, said OMVs comprising a second antigeniccomponent; and 3) mixing the liposomes of (1) with the OMVs of (2). 85.The composition of claim 84, obtainable by mixing the liposomes and theOMVs so that they fuse to form hybrid lipid vesicles that comprise boththe first and the second antigenic component.
 86. A pharmaceuticalcomposition comprising the composition of claim 83 plus apharmaceutically acceptable carrier.
 87. A pharmaceutical compositioncomprising the composition of claim 84 plus a pharmaceuticallyacceptable carrier.
 88. A method of preparing a composition,comprising: 1) obtaining a first composition which contains firstvesicles, said first vesicles having a first antigenic component; 2)obtaining a second composition which contains second vesicles, saidsecond vesicles being OMVs from Gram negative bacteria and having asecond antigenic component different from the first antigenic component;and 3) combining the first and second compositions so as to obtain athird-vesicles-containing composition, wherein thethird-vesicles-containing composition comprises third vesicles havingboth the first antigenic component and the second antigenic component.89. The method of claim 88, comprising separating the third vesiclesfrom the third-vesicles-containing composition, to obtain a purifiedpreparation of the third vesicles.
 90. The method of claim 88, whereinthe first vesicles are liposomes or OMVs.
 91. The method of claim 90,wherein the OMVs are from Neisseria.
 92. A method of preparing acomposition, comprising: 1) obtaining a first composition which containsfirst vesicles, said vesicles being OMVs from Gram negative bacteria andhaving an antigenic component; 2) obtaining a second composition whichcontains second vesicles, said second vesicles comprising a soluble,biologically active component within aqueous cores of the vesicles; and3) combining the first and second compositions so as to obtain a thirdvesicles containing composition, wherein the third vesicles containingcomposition comprises third vesicles which both comprise the antigeniccomponent and also contain, within an aqueous core, the soluble,biologically active component.
 93. The method of claim 92, comprisingseparating the third vesicles from the third-vesicles-containingcomposition, to obtain a purified preparation of the third vesicles. 94.The method of claim 92 wherein the first vesicles are from Neisseria.95. The method of claim 92 wherein the second vesicles are liposomes.96. A composition obtainable according to the method of claim
 88. 97. Acomposition obtainable according to the method of claim
 92. 98. Apharmaceutical composition comprising the composition of claim 96 pluspharmaceutically acceptable carrier.
 99. A pharmaceutical compositioncomprising the composition of claim 97 plus a pharmaceuticallyacceptable carrier.
 100. The composition of claim 84, wherein saidmixture is subjected to an energetic combination step.